A Deeper Look into Antidepressant Dependency: An Investigation on the Hepatic Metabolic Processes Behind Enzyme Cytochrome P450 2C19

1. An Investigation on the Hepatic Metabolic
Processes Behind Enzyme Cytochrome P450 2C19
JAELYN RODRIGUEZ

2. Why Would Research on
Antidepressants Matter to You?

4. A superfamily of enzymes found in ALL
kingdoms of life, and there are over 300,000
distinct CYP proteins in mammals alone.
In humans, these proteins are found in the
inner membrane of the mitochondria or the
endoplasmic reticulum of cells.
.

5. Both the genes encoding
P450 enzymes and the
enzymes themselves
are designated with the
CYP root symbol for the
superfamily.
P450 Enzyme
Enzyme Active Site

6. CYPs and their genes have an
incredible impact on how we
function every single day.
From estrogen and
testosterone to vitamin D, to
possible toxins in the liver
(medicines!)
These proteins oxidize steroids, fatty
acids, synthesize hormones, and
metabolize thousands of modern drugs,
or substrates.
Drug Molecule
Camphor, representing the drug
molecule about to be oxidized by
the CYP active site.

7. "Chemistry is the science of substances,
their properties, structure, and
transformations" - P. Atkins
Antidepressant
Cardiovascular
Antihyperlipidemic /
Antidiabetic
Other (Respiratory,
Gastro, CNS)
Growth Hormone
(Bcps, n.d.)

8. (Bcps, n.d.)
Through the study of antidepressants
and the enzymes that metabolize them,
we can learn so much about the way
our bodies handle medicine.

9. "Chemistry is the science of substances,
their properties, structure, and
transformations" - P. Atkins
Known as the brand name “Lexapro.”
An antidepressant used to treat
major depressive disorder (MDD)
and generalized anxiety disorder
(GAD).
It exists under the subcategory of
antidepressants called Selective
Serotonin Reuptake Inhibitors, or
SSRIs.
National Library of Medicine. (n.d.)

10. "Chemistry is the science of substances,
their properties, structure, and
transformations" - P. Atkins
MDD and GAD symptoms are created from a chemical level.
They begin with a serotonin imbalance, caused by a misuse of
the reuptake mechanism in the nerve synapse.
People with these symptoms physically cannot take in the
correct amount of serotonin, no matter how hard they try.
By ingesting the antidepressant, the SSRI travels to the brain to
stop reuptake of serotonin hormones, and allows them to
travel across the synapse and across the neural network.

11. "Chemistry is the science of substances,
their properties, structure, and
transformations" - P. Atkins
Many studies have
been done on the
effectiveness of
escitalopram on the
brain in normal
conditions.
But not many weigh in
the symptoms people
with MDD and GAD
have, and how that can
greatly change how our
medicines work.
There is significant
confirmation for direct
comorbidity of alcohol
use disorders (AUD)
and MDD and GAD.
People with these
disorders are incredibly
susceptible to alcohol
addiction.
(Kuria et al., 2012; Alcohol Metabolism | NIAAA, n.d.; Anker & Kushner 2019.)
Excessive alcohol use
can lead to a temporary,
but significant change in
the liver microsomes
pH.
With what we know
about enzymes and
denaturing, how will
alcohol change how
these CYP enzymes
work?

12. "Chemistry is the science of substances,
their properties, structure, and
transformations" - P. Atkins
This project will be studying the processes before the medicine gets to the brain.
All possible toxins run through the liver before passing through the body, and
thousands of our CYPs exist and run here.

14. Can a recurring
addiction to alcohol
change hepatic pH,
one so unideal for
CYP2C19 that it
becomes
denatured?

15. In this study, acetaldehyde (CH3CHO) will be used to simulate these pH changes
found in the liver.
Acetaldehyde is created when P450 liver enzymes break down alcohol and is only
active after a person has consumed a large amount of alcohol, indicative of
alcohol addiction or misuse.
This is the stage of alcohol that will be
used in the following experiment.

17. XenoTech has developed an extensive and incredibly well-
manicured assay kit for the 15 most prevalent enzymes in
P450.
The following is an in vitro experiment utilizing human liver
microsomes, so one must acknowledge the difficulty in both
expenses and scientific paperwork regarding the responsibility
of owning human-sourced samples.
I will outline the experiment following this model while taking
note of the limitations of the scope of our course and the
resources of our current lab.

18. Conditions
Liver microsomes are thawed
at ~4℃, and the experiment is
conducted at 37℃.
The substrate (0.1mg of
escitalopram) is dissolved
into a small amount (less than
µL/mL) of organic solvent,
diluted with a
buffer/MgCl2/EDTA solution.
The last three components
are added as a single solution
to the tube.
Control
The basal conditions
described on the left are the
conditions for the control test
tube. Absence of any forms of
alcohol is the control
condition.
Extraction of Results
Incubations of the tubes are
stopped with a denaturant
(typically acid).
The tube is put in a bench-top
centrifuge, and the
precipitated protein is
pelleted after several rounds
of centrifuging.
The resulting protein is
studied for mass, and the
leftover supernatant is
analyzed for any leftover
substrate.
Interpreting
If there is any significant
amount of pelleted protein,
acetaldehyde did not
denature CYP2C19, and an
SSRI protein would be
produced to allow for
serotonin transfer, disproving
the hypothesis.
If there is a large amount of
substrate remaining in the
supernatant, the opposite
would likely be true, leaving a
proven hypothesis.
Trials
Acetaldehyde is added in
varying amounts proportional
to the increasing severity of
alcoholism. (Likely 0.01mL,
0.05mL, 0.1mL, 0.5mL, 1mL)

20. With a positive correlation of acetaldehyde and
CYP2C19 denaturing, results reflect the chemical,
and molecular level effects of incredibly common
symptoms of major depressive disorder and GAD.
The effects of alcoholism intertwine directly with the
success of one’s antidepressant medicine. If one
does not understand the impacts of alcohol on the
effectiveness of their medicine, it can cause
reactions to take more to “feel” like the drug is
working - leading to deeply unhealthy, harmful
tolerance to higher and higher doses of the drug.
This conclusion can affect millions of people taking
escitalopram with a comorbid alcohol addiction.
The impact of this study
reflects much more than
simply the performance of a
singular enzyme in various pH
environments.

22. Alcohol metabolism | National Institute on Alcohol Abuse and Alcoholism (NIAAA).
(n.d.). https://www.niaaa.nih.gov/publications/alcohol-
metabolism#:~:text=First%2C%20ADH%20metabolizes%20alcohol%20to,toxic
%20substance%20and%20known%20carcinogen.&text=Then%2C%20acetalde
hyde%20is%20further%20metabolized,carbon%20dioxide%20for%20easy%20e
limination.
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